Discovery and utilization of sorghum genes (ma5/ma6)

ABSTRACT

Methods and composition for the production of non-flowering or late flowering  sorghum  hybrid. For example, in certain aspects methods for use of molecular markers that constitute the Ma5/Ma6 pathway to modulate photoperiod sensitivity are described. The invention allows the production of plants having improved productivity and biomass generation.

This application claims priority to U.S. Provisional Application No.61/082,388, filed on Jul. 21, 2008. The foregoing application isincorporated herein by reference in its entirety.

This invention was made with government support under grant numberDBI-0321578 awarded by the U.S. National Science Foundation and grantnumber DE-FG02-06ER64306 awarded by the U.S. Department of Energy. TheUnited States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of plant geneticsand molecular biology. More particularly, it concerns producing highbiomass sorghum hybrids by utilizing molecular markers.

2. Description of Related Art

Biomass yield is one of the most important attributes of a biomass orbioenergy crop designed for ligno-cellulosic conversion to biofuels orbioenergy. Growth duration is a primary determinant of biomass yield,therefore late or non-flowering plants accumulate the most biomassassuming environmental conditions allow yield potential to be expressed.

Once grain sorghum initiates flowering, growth of the vegetative plant(stem, leaves) stops so that carbon and nitrogen compounds to be usedfor grain production. As a consequence, biomass accumulation overalldecreases to some extent during the reproductive phase and ceases oncegrain filling has been completed (unless ratooning follows grainproduction).

In contrast, a late or non-flowering bioenergy sorghum crop grown forbiomass production will continue to accumulate biomass by buildinglarger vegetative plants until frost or adverse environmental conditionsinhibit photosynthesis (e.g., drought, cold). It is estimated thatlate/non-flowering biomass sorghum will generate more than two times thebiomass accumulated by grain sorghum per acre assuming reasonable growthconditions throughout the growing season. Therefore, there is a need forproducing late or non-flowering sorghum.

SUMMARY OF THE INVENTION

The present invention overcomes a major deficiency in the art inproducing high biomass sorghum hybrid by using molecular markers forselection.

In one aspect the invention provides a method for producing a lateflowering or non-flowering hybrid sorghum plant comprising crossing afirst early flowering sorghum plant with a second early floweringsorghum plant, wherein each of the first and second early floweringsorghum plants is homozygous recessive for at least one allelecontributing to an early flowering phenotype, and wherein the first andsecond early flowering sorghum plants are not homozygous recessive forthe same allele contributing to an early flowering phenotype. In oneembodiment, the hybrid progeny plant comprises a dominant Ma7 or Ma3allele.

In a further aspect, the invention provides crossing a first sorghumplant heterozygous dominant for at least a Ma5 or Ma6 allele with asecond sorghum plant homozygous recessive for at least the Ma5 or Ma6allele.

In yet a further aspect the invention provides crossing a first sorghumplant homozygous dominant for at least a Ma5 or Ma6 allele with a secondsorghum plant homozygous recessive for at least the Ma5 or Ma6 allele.

In certain embodiments, the first or second early flowering sorghumplant may be produced by a) crossing a late flowering or non-floweringsorghum plant homozygous dominant for Ma5 and Ma6 comprising superiorbioenergy properties with an early flowering sorghum plant homozygousrecessive for a Ma5 or Ma6 allele; b) inbreeding a F₁ progeny; and c)selecting for an early flowering sorghum F₂ plant homozygous recessivefor Ma5 or Ma6 but not homozygous recessive for the same Ma5 or Ma6allele and comprising said superior bioenergy properties.

In another embodiment, the first or second early flowering sorghum plantmay be produced by mutagenizing a late flowering or non-floweringsorghum plant to produce early flowering progeny comprising an inactivegene in a photoperiod sensing pathway. The gene in a photoperiod sensingpathway is selected from the group consisting of Ma3, Ma5, Ma6 and Ma7,in certain embodiments. For instance, the Ma3 gene may comprise anucleic acid encoding PhyB, the Ma5 gene may comprise a nucleic acidencoding a COP9FUS5 homolog or a Myb-transcription factor, the Ma6 genemay comprise a nucleic acid encoding sorghum Prr37, and the Ma7 gene maycomprise a nucleic acid encoding a polypeptide selected from the groupconsisting of PhyC, a MADS-box 14 protein and AP1.

In some embodiments, the first or second early flowering sorghum isselected from the group consisting of ATx623, EBA-3 and R.07007.

In certain embodiments the invention provides a method of selecting fora progeny plant of the cross according to the invention comprisingmarker-assisted selection comprising at least a first genetic markergenetically linked to a Ma5 or Ma6 allele. For instance, in oneembodiment, the genetic marker genetically linked to the Ma5 allele maycomprises a nucleic acid encoding a polypeptide selected from the groupconsisting of COP9FUS5 homolog and a Myb-transcription factor and inanother embodiment, the genetic marker genetically liked to the Ma6allele comprises a nucleic acid encoding a sorghum Prr37. The sorghumPrr37 polypeptide may comprises a lysine at position 166 or may beencoded by a nucleic acid molecule comprising SEQ ID NO:1, in particularembodiments of the invention.

In further embodiments, genetic markers in accordance with the inventionmay be linked to a quantitative trait locus (QTL). In some embodiments,the QTL is selected from the group consisting of FlrAvgB1, FlrAvgD1,FlrFstG1, FltQTL-DFG, FltQTL-DFB, QMa50.txs-A, QMa50.txs-C,QMa50.txs-F1, QMa50.txs-F2, QMa50.txs-H, QMa50.txs-I, QMa1.uga-G,QMa1.uga-D, and QMa5.uga-D.

In still further embodiments, genetic markers in accordance with theinvention may be selected from the group consisting of sequence variantsrevealed by direct sequence analysis, restriction fragment lengthpolymorphisms (RFLP), isozyme markers, allele specific hybridization(ASH), amplified variable sequences of plant genome, self-sustainedsequence replication, simple sequence repeat (SSR) and arbitraryfragment length polymorphisms (AFLP).

Another aspect of the invention provides harvesting a progeny hybridplant of the invention to produce biomass, bioenergy, bioproducts orsugar/starch. In yet another aspect, the invention provides a lateflowering or non-flowering sorghum hybrid seed produced in accordancewith the invention and sorghum hybrid plants grown from the seed.

In a further aspect, the invention provides a method of producing aninbred early flowering sorghum plant comprising: a) crossing a lateflowering or non-flowering sorghum plant homozygous dominant for Ma5 andMa6 with an early flowering sorghum plant homozygous recessive for a Ma5or Ma6 allele; b) inbreeding the F₁ progeny; and c) selecting for anearly flowering sorghum F₂ plant homozygous recessive for Ma5 or Ma6 butnot homozygous recessive for the same Ma5 or Ma6 allele. In certainembodiments, the late flowering or non-flowering sorghum plant comprisessuperior bioenergy properties. In further embodiments, the selectedearly flowering sorghum F₂ plant comprises said superior bioenergyproperties.

In yet a further aspect, the invention provides an inbreed earlyflowering sorghum seed produced in accordance with the invention andinbreed sorghum plants grown from the seed.

In certain aspects, the invention provides a method of identifying thegenotype of a sorghum plant for a Ma5 or Ma6 allele comprising: a)obtaining a sorghum plant; and b) assaying the sorghum plant for agenetic marker genetically linked to the Ma5 or Ma6 allele. In oneembodiment, the genetic marker genetically linked to the Ma5 allele maybe a nucleic acid encoding a polypeptide selected from the groupconsisting of COP9FUS5 homolog and a Myb-transcription factor. Inanother embodiment, the genetic marker genetically linked to an Ma6allele may be a nucleic acid encoding a sorghum Prr37 polypeptide. Incertain embodiments, the sorghum Prr37 polypeptide may comprise a lysineat position 166 or may be encoded by a nucleic acid molecule comprisingSEQ ID NO:1.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan however these terms may be used interchangeably with“comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-C: Alignment of EBA3 and RTx436 SbPRR37 cDNA sequences showingDNA sequence differences (bolded).

FIG. 2: Comparison of protein sequences of Prr37 proteins encoded byEBA-3 and RTx436 derived from cDNA sequences. 2.1 corresponds to thesorghum Prr37 protein encoded by EBA-3 (Ma6) and 2.2 corresponds to thesorghum Prr37 protein encoded by RTx436 (ma6). An amino acid differencein the Prr37 protein putative dimerization domain is bolded andunderlined; (K (lysine) in EBA-3, N (asparagine) in RTx436. Threeadditional differences in amino acid sequence are bolded.

FIG. 3: Alignment of partial promoter sequences of SbPRR37 derived fromEBA-3 and RTx436. Query refers to EBA3 and Subject refers to BTx623.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. THE PRESENT INVENTION

The instant invention overcomes several major problems with currentsorghum production technologies in producing sorghum hybrids that havelong duration of vegetative growth due to late flowering or lack offlowering, from inbreds that will flower sufficiently early in regionsoptimal for hybrid seed production, by manipulation of several QTL andthe corresponding genes/alleles that constitute the Ma5/Ma6 pathway thatregulates photoperiod sensitivity and flowering time in sorghum. Furtherembodiments and advantages of the invention are described below.

II. SORGHUM

Increased demands on the agricultural and forestry industries due toworld population growth, especially recent urgent need in biofuelsproduction, have resulted in efforts to increase plant production and/orsize. Sorghum has been an excellent biomass source with its high yieldpotential, high water use efficiency, and established productionsystems. Certain embodiments of the present invention disclose methodsto generate sorghum genotypes with the genetic potential for improvedbiomass production.

Sorghum is a genus of numerous species of grasses, some of which areraised for grain and many of which are used as fodder plants eithercultivated or as part of pasture. The plants are cultivated in warmerclimates worldwide. Species are native to tropical and subtropicalregions of all continents in addition to Oceania and Australasia.Sorghum is in the subfamily Panicoideae and the tribe Andropogoneae (thetribe of big bluestem and sugar cane). Sorghum is known as great milletand guinea corn in West Africa, kafir corn in South Africa, dura inSudan, mtama in eastern Africa, jowar in India and kaoliang in China.

Sorghum is well adapted to growth in hot, arid or semi-arid areas. Themany subspecies are divided into four groups—grain sorghums (such asmilo), grass sorghums (for pasture and hay), sweet sorghums (formerlycalled “Guinea corn”, used to produce sorghum syrups), and broom corn(for brooms and brushes). The name “sweet sorghum” is used to identifyvarieties of Sorghum bicolor that are sweet and juicy. High biomassSorghum as a source of biofuels has also drawn a lot of attentionrecently.

Sorghum species contemplated in this invention include, but are notlimited to, Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghumarundinaceum, Sorghum bicolor (primary cultivated species), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor,Sorghum virgatum, and Sorghum vulgare.

III. PHOTOPERIOD SENSITIVITY

The present invention relates to methods of modulating photoperiodsensitivity and flowering time in sorghum for high biomass production.Photoperiod sensitivity refers to the fact that some plants will notflower until they are exposed to day lengths that are less than acritical photoperiod (short day plants) or greater than a criticalphotoperiod (long day plants). Long day (LD) and short day (SD) plantdesignations refer to the day length required to induce flowering.Facultative LD or SD plants are those that show accelerated flowering inLD or SD but will eventually flower regardless of photoperiod. Mostplants including sorghum must pass through a juvenile stage (lasting˜14-21 days for sorghum) before they become sensitive to photoperiod.

Sorghum is a facultative SD plant where long days inhibit flowering andshort days accelerate flowering. The degree of photoperiod sensitivityin sorghum refers to the length of the short days that are required toinduce flowering. A highly photoperiod sensitive sorghum will requirephotoperiods less than ˜12 hours before flowering occurs whereas plantswith low photoperiod sensitivity only require day lengths less than ˜14hours to induce flowering. Different sorghum genotypes vary in theirdegree of photoperiod sensitivity. Sorghum inbreds have been identifiedwith photoperiod sensitivity ranging from ˜10.5 to ˜14 hours and stillothers that are nearly completely insensitive to photoperiod. Forexample, in College Station, Tex., photoperiod insensitive sorghumplanted in April will flower in approximately 48-55 days. In contrast,highly photoperiod sensitive sorghum hybrids with the Ma5/Ma6 genotypeflower in mid to late September in College Station, Tex. (˜175-180days).

For example, “early flowering sorghum” may be a plant that flowers in 50to 120 days after planting between April 1 and April 19 in CollegeStation, Tex.; a “late or non-flowering sorghum” may be a plant thatflowers 150 to 200 or more days after planting or does not flower underthese conditions The number of days to flowering will depend on theplanting date and latitude where a sorghum genotype is planted becausethese factors determine when the plants are exposed to days that aresufficiently short to induce flowering. In general, late floweringphotoperiod sensitive plants such as sorghum with the genotype Ma5_Ma6_will not flower until day lengths are less than 12.2 hrs, whereas lessphotoperiod sensitive early flowering sorghum with recessive forms ofMa5, and Ma6 (and potentially Ma 7, Ma3, Ma1, etc.) will flower whenexposed to day lengths (photoperiods) of ˜12.4-14 hr or longer dependingon genotype.

A. Utility of the Ma5/Ma6 System for Bioenergy Sorghum Hybrid Production

Certain aspects of this invention involve the use of the Ma5/Ma6 systemto produce early flowering inbreds that when crossed generate highbiomass or bioenergy sorghum hybrid seed that can be planted at any timeof the year suitable for production, where the hybrid plants will havelong growth duration (i.e., late flowering or non-flowering) at alllatitudes from ˜40 degrees N/S to the equator (40N=the upper mid-westwhere sorghum growth is limited by cold). In another aspect, this sameflowering control system can also be used to design sweet sorghumhybrids that grow for a specified number of days prior to flowering atdifferent latitudes from early flowering inbreds suitable for hybridseed production.

Table 1 below describes the relationship between latitude and daylengthat planting and harvest for biomass/bioenergy production regions from˜40 degrees N/S to the equator. At higher latitudes, planting date islater in the year and harvesting occurs earlier due to longer durationof winter and low temperatures (shorter season). At lower latitudes,planting can be done earlier in the year or virtually any time in somelocations and harvesting later in the year or multiple times during theyear, including times of the year when daylength is less than 12 hours(Table 1).

TABLE 1 Relationship between latitude of crop production and daylengthPlanting Daylength Harvest Daylength City Latitude date hours date hoursDes Moines, IA 41.35 N 15-May 14.3 1-Oct 11.6 New York, NY 40.42 N30-May 14.6 1-Oct 11.6 Amarillo, TX 35.05 N 15-May 13.8 15-Oct 11.1College Station, TX 30.37 N 20-Mar 11.7 15-Nov 10.4 Beaumont, TX 30.05 N20-Mar 11.8 15-Nov 10.5 Weslaco, TX 26.09 N 20-Mar 11.8 1-Dec 10.5Puerto Rico 18.57 N monthly 10.8-13.2 monthly 10.8-13.2 Panama City08.57 N monthly 11.4-12.6 monthly 11.4-12.6 Equator 0 monthly 12  monthly 12   Brazilia, Brazil 16.12 S monthly 11.1-12.9 monthly11.1-12.9 Brisbane, AU 27.30 S 20-Sep 11.9 15-Mar 12.2

Sorghum is insensitive to photoperiod during the juvenile phase whichlasts for ˜14-21 days post planting depending on genotype. Therefore,bioenergy sorghum hybrids need to have sufficient photoperiodsensitivity to prevent flowering at the daylengths that occur ˜14-21days post-planting at all latitudes used for bioenergy crop production.In addition, bioenergy sorghum hybrids that are planted in long daysthat block flowering may also require increased photoperiod sensitivityin order to block flowering prior to frost or harvest if daylengthsdecrease significantly during the growing season.

Certain aspects of the present invention involves the identification ofallelic combinations of Ma5/Ma6 and other genes that repress floweringthat work in hybrid combination to block flowering at daylengths asshort as 11-10.5 hours. The early flowering inbreds used to producelate/non-flowering hybrid seed are designed to flower early due todifferent recessive genes that control flowering time. Therefore, whenthese inbreds are crossed, the F1 hybrids contain dominant genes at allloci involved thereby delaying flowering until plants are exposed tovery short photoperiods.

Certain embodiments of the present invention provide sorghum genotypesthat contain versions of Ma5 and Ma6 that in combination delay floweringuntil day lengths are less than 12 hr 20 min (Rooney and Aydin, 1999).There is evidence that additional genes such as Ma1-Ma4 enhance sorghumphotoperiod sensitivity. In addition, it is likely that differentalleles of Ma5 and Ma6 exist that can be used to make bioenergy sorghumhybrids even more photoperiod sensitive (less than 12 hr) increasingtheir utility for growing regions closer to the equator where bioenergysorghum will be planted and grown in day lengths shorter than 12 hours(Table 1). For example, a study by Miller et al. (1968) identified fivegroups of sorghum that had short day requirements for flowering thatranged from ˜13 hr to ˜11.1 hr. This genetic material, and othergenotypes identified in accordance with the present invention, flowerlate when growing at low latitudes in places such as Puerto Rico. Inanother study, Craufurd et al. (1999) identified sorghum genotypes withcritical photoperiods between 10.2 and 11 hrs. In certain aspects of theinvention, these materials have been investigated to identify genes withsimilar action to Ma5/Ma6 and alleles of Ma5 and Ma6 that would beuseful for breeding PS hybrids for use over the entire range oflatitudes from 40N/S to the equator.

B. Genetic Pathway of Photoperiod Sensitivity and Uses Thereof

Photoperiod sensitivity and late flowering is mediated in sorghum andrice by genes that repress activation of FT (flowering locus T) and AP1and the transition of the apex from vegetative growth to formingreproductive structures. The repressors of flowering in sorghum act in adominant fashion. The repressors are inactivated or less active undershort photoperiods (and thermal periods). The vegetative ornon-flowering state is maintained in part by light mediated signalingthrough PhyB and PhyC and possibly from other sources (PhyA, etc.) andpartly by output from a circadian clock. The light signaling pathwayinvolves a series of steps and genes, some of which may act directly torepress FT, and others of which act downstream from the circadian clockthrough modulation of homologs of GI, CO, and other genes that modulaterepression of FT.

The repressing pathway can be inactivated by disrupting the function ofany of the genes that are in the signaling pathway (PHYB, PHYC, or agene between the photoreceptors and FT, and genes involved in clockfunction or input/output). The disruption of a gene in the flowerrepression pathway converts a photoperiod sensitive genotype into a lessphotoperiod sensitive genotype or photoperiod insensitive genotype thatwill flower early or at longer day lengths. If genotypes that arephotoperiod insensitive due to inactivation of different genes in theflowering repression pathway are crossed, then the hybrid will bephotoperiod sensitive and later flowering because active allelescontributed by the gametes from each line complement inactive allelespresent in the gametes/genome of the other parental inbred line.

IV. PRODUCTION OF PHOTOPERIOD SENSITIVE HYBRID USING MA5/MA6 SYSTEM

In certain aspects of the present invention, early flowering inbredsorghum genotypes with the proper allelic combinations of Ma5 and Ma6can be crossed to produce photoperiod sensitive late-flowering sorghumhybrids (Ma5_Ma6_) ideal for biomass/bioenergy production with the useof molecular markers. In one embodiment, the early flowering photoperiodinsensitive sorghum inbreds contain complementary pairs ofdominant/recessive Ma5/Ma6 genes (Ma5ma6 and ma5Ma6 respectively).

One advantage of the Ma5/Ma6 system is the ability of this system toproduce sorghum hybrids that have long duration of vegetative growth dueto late flowering or lack of flowering, from inbreds that will flowersufficiently early in regions optimal for hybrid seed production (suchas high plains of Texas).

The production of bioenergy sorghum hybrids is also important becausehybrids are preferred commercially due to hybrid vigor that generatesgreater yield, and the ability to better control seed stocks throughhybrid seed production. The increase in yield attributed to hybrid vigorin sorghum is typically ˜20% to ˜50%. Photoperiod sensitive bioenergysorghum hybrids that flower late or that do not flower are important forbioenergy production for several reasons: long duration of vegetativegrowth associated with late/non-flowering genotypes increases biomassyield per acre, high levels of photoperiod sensitivity will allow nearlyyear round planting of bioenergy sorghum hybrids at lower latitudes, andplants growing vegetatively (non-flowering) are more drought tolerantthan plants that are in the reproductive phase of development; this isan important attribute of bioenergy sorghum.

A. Breeding Material and Methods

In further aspects of the present invention, naturally occurring allelesof Ma5 and Ma6 as well as other maturity genes (e.g., PHYB, PHYC) thatare involved in the photoperiod-sensing pathway can be used to constructearly flowering inbreds that can be crossed to produce late floweringhybrids.

In one embodiment, sorghum line R.07007 or EBA-3 is a primary source ofboth ma5 (recessive form) and Ma6 (dominant form), although otherversions of Ma6 derived from photoperiod sensitive sorghum accessionsmay also be utilized. In another embodiment, dominant forms of Ma5 arederived from grain sorghum female lines that may be used for hybrid seedproduction.

In addition to working with naturally occurring genetic variants,certain embodiments of the present invention comprise mutagenizing anygroup of PS genotypes and identify PI lines derived from the parentallines that contain an inactive gene in the pathway that repressesflowering. Crossing photoperiod insensitive early flowering genotypesthat contain different inactive genes in the pathway that controlsflowering time will generate photoperiod sensitive late floweringhybrids.

An exemplary approach involves screening photoperiod sensitive (lateflowering) sorghum germplasm for accessions that express superiorbioenergy traits. These accessions (most likely Ma5/Ma6) are thencrossed to R.07007 or EBA-3 (ma5ma5ma7ma7Ma6Ma6). F2 progeny from thesecrosses that flower early (ma5ma5) but that retain Ma6Ma6 are selectedby phenotyping and marker-assisted selection. The resulting earlyflowering inbreds (ma5ma5Ma6Ma6) can then be crossed with elite grainfemale A-lines that have the genotype (Ma5Ma5Ma7Ma7ma6ma6), to producebioenergy hybrids that are Ma5_Ma7_Ma6_ that will flower late.

B. Sorghum Mutagenesis

In another aspect of the invention, mutagenesis of late floweringsorghum genotypes to create early flowering genotypes could be carriedout in the following exemplary manner. The seed from a late floweringsorghum inbred would be germinated and treated with a mutagen such asEMS (ethyl methanesulphonate) or ENU (1-ethyl-1-nitrosourea) or usingX-rays or neutron bombardment to induce changes in DNA sequencethroughout the sorghum genomes of thousands of seedlings. The M1seedlings (M1 refers to the first generation of plants that were exposedto a mutagen) surviving the treatment would be grown to maturity andself-pollinated. M2 seed derived from a large number of M1 plants wouldbe grown out and screened for M2 plants that flower early underconditions where the parental inbred flowers late. An early floweringphenotype would be consistent with mutation in a gene that repressesflowering such as Ma5 or Ma6.

C. Molecular Markers

a. Marker Assisted Selection

Marker assisted selection or marker aided selection (MAS) is a processwhereby a marker (morphological, biochemical or one based on DNA/RNAvariation) is used for indirect selection of a genetic determinant ordeterminants of a trait of interest (e.g., productivity, diseaseresistance, abiotic stress tolerance, and/or quality). This process hasbeen used in plant breeding.

Considerable developments in biotechnology have led plant breeders todevelop DNA marker aided selection systems to augment traditionalphenotypic-pedigree-based selection systems. Marker assisted selection(MAS) is an indirect selection process where a trait of interest isselected not based on the trait itself but on a marker linked to thegene (allele) that controls expression of the trait. For example if MASis being used to select individuals with disease resistance, then amarker allele which is linked to the gene conferring disease resistanceis scored or selected for rather than disease resistance per se. Theassumption is that the marker allele is associated with the gene and/orquantitative trait locus (QTL) of interest that confers the trait underselection. MAS can be useful to select for traits that are difficult tomeasure, exhibit low heritability, and/or are expressed late indevelopment.

In certain embodiments, a marker may be;

Morphological—First marker loci available that have obvious impact onmorphology of plant. Genes that affect form, coloration, male sterilityor resistance among others have been analyzed in many plant species.Examples of this type of marker may include the presence or absence ofawn, leaf sheath coloration, height, grain color, aroma, etc.

Biochemical—A gene that encodes a protein that can be extracted andobserved; for example, isozymes and storage proteins.

Cytological—The chromosomal banding produced by different stains; forexample, G banding.

Biological—Different pathogen races or insect biotypes based on hostpathogen or host parasite interaction can be used as a marker since thegenetic constitution of an organism can affect its susceptibility topathogens or parasites.

DNA-based and/or molecular—A unique (DNA sequence), occurring inproximity to or within the gene or locus of interest, can be identifiedby a range of molecular techniques such as direct sequencing, RFLPs,RAPDs, AFLP, DAF, SCARs, microsatellites, etc. DNA markers detectvariation in DNA sequence, or DNA polymorphisms, that distinguishindividuals. DNA polymorphisms include differences in single nucleotidesequences (SNPs), simple sequence repeats (SSRs), inversions ordeletions (INDELS). DNA markers are designed to identify DNA sequencedifferences by one of several methods including; direct sequenceanalysis, electrophoretic separation of DNA fragment sizes followingdigestion of genomic DNA with restriction enzymes (RFLP) or after DNAamplification using PCR (AFLP, SSRs), or based on differences inamplification or probe hybridization (microarrays, Taqman probes, etc.).

As used herein, an “inherited genetic marker” is an allele at a singlelocus. A locus is a position on a chromosome, and allele refers toconditions of genes; that is, different nucleotide sequences, at thoseloci. The marker allelic composition of each locus can be eitherhomozygous or heterozygous.

Coinheritance, or “genetic linkage,” of a particular trait and a markersuggests that they are physically close together on the chromosome.Linkage is determined by analyzing the pattern of inheritance of a geneand a marker in a cross. The unit of recombination is the centimorgan(cM). Two markers are one centimorgan apart if they recombine in meiosisonce in every 100 opportunities that they have to do so. The centimorganis a genetic measure, not a physical one. Those markers located lessthen 50 cM from a second locus are said to be genetically linked,because they are not inherited independently of one another. Thus, thepercent of recombination observed between the loci per generation willbe less than 50%. In particular embodiments of the invention, markersmay be used located less than about 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1or less cM apart on a chromosome.

The gene of interest is directly related with production of protein(s)or RNAs (e.g., miRNA) that produce certain phenotypes whereas markersshould not influence the trait of interest but are genetically linked toan allelic form of a gene that modifies a trait (and so the marker andgene remain together during segregation of gametes due to the physicallinkage between marker and gene, and a reduction in homologousrecombination between the marker and gene of interest due to their closeproximity on a strand of DNA). In many traits genes are discovered andcan be directly assayed for their presence with a high level ofconfidence. However, if a gene is not isolated, marker's help is takento tag a gene of interest. In such case there may be some false positiveresults due to recombination between marker of interest and gene (orQTL). A preferred marker that corresponds to or detects the differencein DNA sequence causing the desired phenotype or trait would elicit nofalse positive results.

In MAS, generally the first step is to map the gene or quantitativetrait locus (QTL) of interest first by using one or more genetic mappingtechniques and then use this information to identify DNA markers linkedto and flanking the QTL useful for marker-assisted selection. Generally,the markers to be used should be close to the gene of interest (<5recombination unit or cM) in order to ensure that only a minor fractionof the selected individuals will have a recombination between the DNAmarker and target gene following any given cross or meiosis(specifically the DNA sequence variation within the target gene thatcauses the desired trait). Generally, not only a single marker butrather two markers are used that flank the target gene or QTL as closelyas possible in order to reduce the chances of an error due to homologousrecombination.

In plants QTL mapping is generally achieved using bi-parental crosspopulations involving two parents that have a contrasting phenotype forthe trait of interest. Commonly used populations are recombinant inbredlines (RILs), doubled haploids (DH), back cross and F2. Linkage betweenthe phenotype and markers that have already been mapped is tested inthese populations in order to determine the position of the QTL on theoverall genetic map. Such techniques are based on linkage and aretherefore referred to as “linkage mapping”.

In contrast to two-step QTL mapping and MAS, a single-step method forbreeding typical plant populations has been developed (Rosyara et al.,2007). In such an approach, in the first few breeding cycles, markerslinked to the trait of interest are identified by QTL mapping and laterthe same information in used in the same population. In this approach,pedigree structure is created from families that are created by crossinga number of parents (in three-way or four way crosses). Phenotyping iscarried out and genotyping is done using molecular markers mapped thepossible location of QTL of interest. This will identify markers andtheir favorable alleles. Once these favorable marker alleles areidentified, the frequency of such alleles will be increased and responseto marker-assisted selection is estimated. Marker allele(s) withdesirable effect will be further used in next selection cycle or otherexperiments.

Recently high-throughput genotyping techniques are developed whichallows marker aided screening of many genotypes. This will help breedersin shifting traditional breeding to marker-aided selection. One exampleof such automation is using DNA isolation robots and pipetting robots. Arecent example of a high throughput DNA sequencer is the Illumina SGAIIor ABI SOLID System.

Genetic markers and QTLs used in certain embodiments of the inventionhave been disclosed below.

In certain embodiments of the invention, molecular markers are developedthat are polymorphic in parental lines of a cross or population, andlinked to and flank the Ma5 and Ma6 QTL targeted for marker assistedselection (MAS). The molecular markers in some cases can correspond toand detect specific DNA sequence variants causing dominant or recessivegene action. In certain aspects, DNA may be extracted from the parentalsorghum lines and progeny of a cross (F1, F2, backcross, testcross, RIL,etc.) and analyzed with molecular markers for the presence or absence ofmarker alleles linked to and flanking regions of the genome encodingdominant or recessive forms of Ma5 and/or Ma6. While any molecularmarker assay technology could be used, biallelic (or multiallelic)marker assays such as SSRs, or assays such as direct sequencing thatdetect SNPs/indels are preferred.

b. Ma Genes

There are six classic maturity genes in sorghum that control floweringtime termed Ma1-Ma6. Ma1, Ma2, Ma3 and Ma4 were identified by Quinby andhis colleagues (Quinby and Karper, 1946; Quinby, 1966; Quinby, 1974).These loci/genes are part of a pathway that inhibits flowering.Therefore in general, sorghum plants with recessive Ma1-Ma6 genes (withlow or no activity) flower earlier than plants with dominant or activeMa1-Ma6 genes that repress flowering. Sorghum plants that areMa1Ma2Ma3Ma4 but recessive at either Ma5 or Ma6 will flower in ˜74 daysin College Station, Tex. when planted on April 19 (Rooney and Aydin,1999) or in ˜85 days when planted on June 1 in Plainview, Tex. (Quinby,1974). Plants with recessive genes at Ma1-Ma4 (and recessive at Ma5 orMa6) will flower in ˜48-55 days post planting in these same locations.Ma5 and Ma6 are an additional pair of maturity loci that delay floweringwhen sorghum is planted ˜April 19 in College Station, Tex. for ˜175 days(mid-late September when photoperiods decrease below 12 h 20 min)(Rooney and Aydin, 1999). Based on information described in more detailbelow, it is predicted that late flowering Ma5/Ma6 plants also requirean active PHYB gene (Ma3)

If an active form of PHYB (or Ma3) is required for Ma5/Ma6 genotypes toexpress photoperiod sensitivity and flower late, then complementarydominant/recessive forms of Ma3 could also be used to modulatedifferential flowering time in certain types of inbreds and hybrids. Inthis case, an early flowering inbred sorghum line that has the genotypema3ma3Ma5Ma5Ma6Ma6 could be crossed to a second early flowering inbredsorghum genotype that has the genotype Ma3Ma3Ma5Ma5ma6ma6 in order toproduce late flowering sorghum hybrids with the genotypeMa3ma3Ma5Ma5Ma6ma6.

Table 2 shows that information about the genetic map location of Ma1 andMa3 has been published (Klein et al., 2008; Childs et al., 1997). Ma3encodes the red light photoreceptor phytochrome B that is known tomediate repression of flowering in short day and long day plants (Childset al., 1998). In addition, the inventors have collected informationover the past several years on the genetic map locations of Ma5 and Ma7,loci required in combination with Ma6 to delay flowering ˜175 days inCollege Station. Ma6 has also been mapped, as well as a modifier of Ma6activity.

TABLE 2 Sorghum maturity (Ma) genes Locus Map location Gene ReferenceMa1 SBI06, ~11-21cM Unknown Klein et al., 2008 Ma2 Unknown Ma3 SBI01,~166cM PHYB Childs et al., 1998 Ma4 Unknown Ma5 SBI02, ~145-148cM Ma7SBI01, ~23-26cM Ma6 SBI06, ~11-19cM

b. Sorghum Flowering Time QTL

QTL (quantitative trait loci), quantitative trait inheritance orpolygenic inheritance refers to the inheritance of a trait or phenotypethat varies in degree of trait expression due to the interactionsbetween two or more genes and the environment. QTL are genetic loci thatspan regions of a genome that encode genes that contribute toquantitative inheritance of a trait. The contributions of allelic formsof genes that contribute to quantitative traits and the genetic maplocations of QTL can be characterized by analysis of populations derivedby crossing parental lines that contain different allelic forms of genesthat contribute to quantitative trait inheritance.

QTL mapping involves the genetic study of inheritance of alleles thatoccur in two or more loci and the phenotypes (physical forms or traits)that they produce. Because most traits of interest are governed by morethan one gene, defining and studying the entire suite of genes and theiralleles that modulate a trait provides an understanding of what effectthe genotype of an individual has on the phenotype of that individual.

Genetic analysis involving statistical assessment is required to analyzethe interaction of genes and to determine whether they produce asignificant effect on the phenotype. QTL identify regions of the genomeas containing allelic variation for one or more genes (or regulatoryelements) that modulate the trait being assayed or measured. They areshown as intervals spanning a region of a chromosome, genetic map, orDNA sequence, where the probability of association is plotted for eachmarker used in the mapping experiment.

The QTL techniques were developed in the late 1980s and can be performedon populations of any species. To begin, a set of genetic markers mustbe developed for the species in question. A marker is an identifiableregion of variable DNA sequence (single nucleotide or repeat variation,or inversions/deletions). Biologists are interested in understanding thegenetic basis of phenotypes (i.e., physical traits). The aim is to finda marker that is significantly more likely to co-occur (co-segregatefollowing a cross) with the trait than expected by chance, that is, amarker that has a statistically significant association with the trait.It is ideal to identify the specific gene or genes that modulate thetrait in question, but this often requires a great deal of time andeffort. Instead, they can more readily find regions of DNA that are veryclose to the genes in question. When a QTL is mapped, it identifies aregion of the genome that spans the actual gene underlying thephenotypic trait although the region identified may also encode manygenes that do not modulate the target trait.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is modulated by many independent loci, or by a few loci, anddo those loci interact. This can provide information on how expressionof the phenotype is regulated.

Numerous QTL that modulate flowering time in sorghum have beenidentified in various studies (e.g., Lin et al., 1995, Paterson et al.,1995, Crasta et al., 1999, Hart et al., 2001; Feltus et al., 2006). Thecorrespondence between QTL that modulate flowering time identified ingenetic mapping studies and Ma1-Ma6 is not entirely clear because thelocation of Ma2 and Ma4 on the sorghum genetic map is not known.Information on various QTL for flowering time in sorghum is listed inTable 3.

TABLE 3 Sorghum flowering time QTL Locus Map location Lin et al., 1995,Paterson et al., 1995; BTx623 X S. propinquum Marker FlrAvgB1 SBI02,~102-119cM UMC5, UMC139 FlrAvgD1 SBI06, ~9-21cM FlrFstG1 SBI09,~129-150cM UMC132 Crasta et al., 1999; B35 X RTx430 Gene FltQTL-DFGSBI10, ~70-74cM UMC21 FltQTL-DFB SBI01, ~45cM UMC27, ~10cM from PHYAHart et al., 2001 (see map positions in Feltus et al., 2006 below)Feltus et al., 2006; summary of QTL from BTx623/IS3620C; BTx623/S.propinquum Marker QMa50.txs-A SBI01, ~182-186cM Xgap36 QMa50.txs-CSBI03, ~140cM Xumc16-Xtxs422 QMa50.txs-F1 SBI09, ~143cM Xcdo393QMa50.txs-F2 SBI09, ~143cM Xcdo393 QMa50.txs-H SBI08, ~130-136cMXtxp105-Xtxs1294 QMa50.txs-I SBI06, ~10-36cM Xumc119-Xcdo718 Lin et al.(1995), Paterson et al. (1995) QMa1.uga-G SBI09, ~129-150cMXumc132-pSB445 QMa1.uga-D SBI06, ~31-59cM data requires further analysisQMa5.uga-D SBI06, ~8-20cM tiller flowering

The relationship between Ma6 and Ma1 is uncertain at this time. Theimpact of Ma1 and Ma6 is quite different, but both QTL map to a similarregion on SBI06 making it formally possible that Ma1 and Ma6 aredifferent alleles of the same gene or different genes that reside in thesame region of the genome.

Feltus et al. (2006) reported a flowering time QTL (QMa5.uga-D) thatcontrols tiller flowering time that overlaps the region spanned by Ma1and Ma6. It is formally possible that QMa5.ugaD corresponds to adifferent allele of Ma1 or Ma6 or a different flowering time gene.

Lin et al. (1995) mapped a flowering time QTL (FlrAvgD1=QMa1.ugaD) onSBI06 (31-59 cM) and suggested that this QTL could correspond to Mal.Klein et al. (2008) using genotypes known to segregate for Ma1 showedthat Ma1 mapped to an adjacent region on SBI06 (˜11-21 cM). The data inLin et al. (1995) are inconsistent with the assigned map location ofQMa1.ugaD in Feltus et al. (2006). Data in Lin et al. (1995) show thatQMa1.ugaD maps to the same location as QMa5.ugaD (Feltus et al., 2006).

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods for Using Marker-Assisted Selection of Sorghum Inbredsthat for Production of Photoperiod Sensitive Late or Non-FloweringSorghum Hybrids

In one non-limiting embodiment of the invention, molecular markers maybe used to help convert a photoperiod sensitive (PS) late floweringinbred sorghum (A) that has the genotype Ma5Ma5Ma6Ma6 into a photoperiodinsensitive (PI) early flowering inbred that can be used (crossed) intemperate regions to produce sorghum hybrid seed and hybrids that flowerlate. This can be done as follows:

a. Cross PS sorghum (A) with the genotype Ma5Ma5Ma6Ma6 to a PI sorghum(B) with the genotype ma5ma5Ma6Ma6 to generate an F1 plant.

b. Self the F1 plant and grow out F2 progeny.

c. Use DNA markers to identify progeny (C) that have the genotypema5ma5Ma6Ma6.

d. The ma5ma5 alleles in (C) will be derived from (B).

e. The Ma6Ma6 alleles in (C) may be derived from either (A) or (B).Selection for the source of Ma6 allele may be important depending on therelative activity of the Ma6 alleles derived from (A) and (B).

f. Cross progeny with the genotype ma5ma5Ma6Ma6 (C) to an elite PI earlyflowering sorghum (D) with the genotype Ma5Ma5ma6ma6 to produce F1 seed.

g. F1 hybrid plants derived from this cross will be photoperiodsensitive and late or non-flowering with the genotype Ma5ma5Ma6ma6.

In another aspect, the method described above that starts with PS lateflowering plants with the genotype Ma5Ma5Ma6Ma6 could involve severalalternatives:

a. PI sorghum (B) used above could have the genotype ma5ma5ma6ma6.

In this case, progeny (C) identified by markers with the genotypema5ma5Ma6Ma6 would have derived ma5 alleles from (B) and Ma6 allelesfrom (A).

b. PS sorghum (B) could have the genotype ma5ma5ma7ma7Ma6Ma6.

i. This is a case where recessive alleles in two different genes withMa5-like action are needed to make progeny (C) PI, early flowering, anduseful for the generation of PS sorghum hybrids.

ii. In this case, DNA markers would be used to identify progeny (C) thathave the genotype ma5ma5ma7ma7Ma6Ma6.

iii. In this case, PI progeny (C) with the genotype ma5ma5ma7ma7Ma6Ma6could be crossed to an elite PI line (D) with the genotypeMa5Ma5Ma7Ma7ma6ma6 to produce PS late or non-flowering sorghum hybridseed/plants with the genotype Ma5ma5Ma7ma7Ma6ma6.

In a further aspects, inventors may want to convert a PI early floweringplant that is not suitable for use in the production of PS late ornon-flowering sorghum hybrids into a PI early flowering plant that canbe used for this purpose. This can be done as follows:

a. Cross a PI early flowering genotype (E) with the genotypema5ma5ma6ma6 or Ma5Ma5ma6ma6 with a PI early flowering genotype (F) withthe genotype ma5ma5Ma6Ma6.

b. Self the resulting F1 plants and use molecular markers to identifyprogeny (G) with the following genotype; ma5ma5Ma6Ma6.

c. The ma5ma5 alleles could be derived from (E) or (F) depending on thecross involved, whereas the Ma6Ma6 alleles will be derived from (F).

d. Cross progeny (G) to an elite sorghum with the genotype Ma5Ma5ma6ma6to generate F1 seed/hybrid plants that are PS late or non-flowering.

Example 2 Genetic Map Analysis of Ma5

The location of Ma5, Ma7 and Ma6 on the sorghum genetic map wasdetermined as described below thus enabling the development of DNAmarkers flanking these loci for use in marker-assisted breeding. Thecoordinates of Ma5, Ma7 and Ma6 on the TAMU sorghum genetic map (cM) andon the DOE sorghum genome sequence (bp) are listed below: (i) Ma5 QTLcoordinates on SBI-02: From 59L10, 67923811 bp, 146.1-148.9 cM totxp428, 68393290 bp, 148.9-152.1 cM; (ii) Ma7 QTL coordinates on SBI-01:From txp208, 6545866 bp, 23.4 cM to txp523, 8017655 bp, 26.5-29.5 cM.

Populations segregating for Ma5/ma5 were constructed by crossing EBA-3(ma5ma5Ma6Ma6) to A3RTx436 (Ma5Ma5ma6ma6) creating an F1 hybrid that wasbackcrossed to EBA-3 to create a BC1F1 mapping population that wasexpected to segregate 1:1 for alleles at the Ma5 locus (Ma5ma5Ma6_;ma5ma5Ma6_). Phenotypic analysis of flowering time of BC1F1 progeny wasperformed from this cross.

A large population of ˜4200 BC1F1 plants was grown at two locations inCollege Station, Tex. and was assayed for days to flowering atapproximately weekly intervals. The parents of this population floweredbetween 60-90 days, and the F1 flowered at ˜170-180 days. Data on timeto flowering was collected from 2915 plants, whereas the remainingplants either died during growth (a small number of plants) or had notflowered by November when frost terminated their development.Approximately 28% of the population flowered early (before August 7) andthere was a period from 105-148 days post planting where fewer plantsflowered before a second large cohort of plants initiated flowering.Approximately 72% of the plants flowered after August 7, with manyplants flowering well after F1 hybrids flowered at approximately 175days (more than 220 days). This result indicated that more than one genewith Ma5-like action was segregating in this population based ondeviation from 1:1 segregation of PI:PS phenotypes and transgressivesegregation for late flowering.

A form of bulk segregant analysis and SSR and AFLP markers were used tomap the location of one locus with Ma5 action to a ˜10 cM region onLG-02. This locus was designated Ma5. The location of Ma5 was furtherrefined to a region ˜250,000 bp. Information on the segregation of Ma5and flowering phenotypes was used to map a second locus with Ma5 likeaction to LG-01. This locus was designated Ma7. The gene for Ma7 wasfurther fine mapped to a region spanning ˜400,000 bp. Portions of thesorghum genome sequence released by DOE to each of these regions andidentified putative genes encoded by these regions based on BLASTanalysis and comparison to the colinear region of the rice genome wereidentified and aligned.

The Ma5 and Ma7 loci were examined and candidate genes in these regionswere identified that could explain the observed regulation of floweringtime. In the Ma5 locus, a gene homologous to COP9FUS5 was identified asa candidate gene. COP9FUS5 is a subunit of a large signalasome complex(CSN complex) that was initially identified in Arabidopsis as involvedin the repression of photomorphogenesis and a range of other activities.This complex acts by targeting transcription factors for degradationthat mediate light activated events (such as de-etiolation, lightactivated gene expression). Therefore, it was reasoned that variation inthe activity of COP9FUS5 could modulate flowering time in sorghum bymodulating light dependent repression of flowering mediated by the PhyBand PhyC photoreceptors, or by modulating light dependent output fromthe circadian clock. A gene encoding a Myb-transcription factor thatcould be involved in flowering time was also identified in the Ma5 finemapping interval. Myb-transcription factors such as CCA1/LHY(Arabidopsis) are a central part of the circadian clock and allelicvariation in this type of gene can modulate flowering time.

Several candidate genes were identified in the Ma7 locus including PhyC,a MADS-box 14 gene and a MADS-box gene corresponding to API. APIactivates meristem identity genes that are involved in the production offloral organs. API is activated by FT in the apex. FT encodes atransmissible protein that travels from the leaf to the apex whenphotoperiod and other requirements are met such that FT expression isactivated. MADS-box 14 is involved in flowering time control in rice soit is also a candidate gene for Ma7. PhyC is also a reasonable candidatefor Ma7 because in rice, and presumably sorghum, inactivation of PhyCdecreases repression of flowering in long days resulting in earlyflowering (as observed in EBA-3).

Example 3 Genetic Map Location and Molecular Description of Ma6

Ma6 was mapped in a BC1F1 population created by crossing EBA-3(ma5ma5Ma6Ma6) to ATx623 (Ma5Ma5ma6ma6), where the F1 derived from thiscross was backcrossed to ATx623. Progeny from the BC1F1 population wereexpected to segregate for the Ma6 locus in a 1:1 ratio (Ma5_ma6ma6 vs.Ma5_Ma6ma6). Late flowering plants from this population were expected tocontain the EBA-3 dominant version of Ma6. Genetic mapping initiallylocated Ma6 to an interval on SBI06 spanning from ˜8 cM to ˜21 cM (Ma6QTL coordinates on LG-06: txp658, 39379760 bp, 8.0-9.9 cM to txp434,42610705 bp, 17.4-20.7 cM; bp coordinates are derived from the DOEpseudomolecule sequence). Further fine mapping narrowed the Ma6 locus toa region spanning from Xtxp598 to a DNA polymorphism present in a DNAbinding protein upstream from Ma6. There are approximately ˜20 annotatedgenes (excluding genes associated with transposons) in this delimitedregion.

A sorghum gene encoding a homolog of Arabidopsis PRR7 (and rice OsPRR37)was present among the ˜20 genes in the delimited Ma6 locus. ThePRR7/PRR37 gene homologs are known to modulate flowering time in severalplant species (Arabidopsis, rice, barley) suggesting that the sorghumPRR7/37 gene homolog in the Ma6 locus is likely to be the gene causingdifferences in flowering time in sorghum. Therefore, cDNA derived fromthis gene was sequenced from EBA-3 (Ma6) and RTx436 (ma6) and compared(FIG. 1A-C; SEQ ID NOs: 1 and 2). The alignment of the cDNA sequencesrevealed 5 sequence polymorphisms (FIG. 1A-C). One of these sequencedifferences (a G (EBA3) to T (RTx436) substitution) caused amino acid166 to change from a lysine (EBA3) to an asparagine in RTx436 (FIG. 2).This amino acid change is conservative (no charge change) but occurs ina three amino acid sequence of the Prr protein predicted to be involvedin dimerization. Therefore it is possible that this change in amino acidsequence alters protein-protein interaction required for normal functionof the SbPrr37 protein.

The protein encoded by sorghum PRR37 (Ma6) is homologous to and similarin amino acid sequence to the protein encoded by rice PRR37. The ricePrr37 protein sequence is shown below, where the putative signalreceiver domain is shown in bold and the putative dimerization domain(amino acids 166-168) is shown in bold and underlined (amino acidsequence KPI (lysine-proline-isoleucine). The sorghum Prr37 putativedimerization domain of EBA-3 (Ma6) has the sequence KPI (SEQ ID NO:3)identical to rice Prr37 (SEQ ID NO:5) (KPI at positions 166-168),whereas the putative dimerization domain of Prr37 from RTx436 (ma6) (SEQID NO:4) has the sequence NPI.

Rice Prr7 Sequence:

1 mmgtahhnqt agsalgvgvg dandavpgag gggysdpdgg pisgvqrppq vcwerfiqkk 61tikvllvdsd dstrqvvsal lrhcmyevip aengqqawty ledmqnsidl vltevvmpgv 121sgisllsrim nhnicknipv immssndamg tvfkclskga vdflv kpi rk nelknlwqhv 181wrrchsssgs gsesgiqtqk caksksgdes nnnngsnddd dddgvimgln ardgsdngsg 241tqaqsswtkr aveidspqam spdqladppd stcaqvihlk sdicsnrwlp ctsnknskkq 301ketnddfkgk dleigsprnl ntayqsspne rsikptdrrn eyplqnnske aamenleess 361vraadligsm aknmdaqqaa raanapncss kvpegkdknr dnimpslels lkrsrstgdg 421anaiqeeqrn vlrrsdlsaf tryhtpvasn qggtgfmgsc slhdnsseam ktdsaynmks 481nsdaapikqg sngssnnndm gsttknvvtk pstnkervms psavkanght safhpaqhwt 541spanttgkek tdevannaak raqpgevqsn lvqhprpilh yvhfdvsren ggsgapqcgs 601snvfdppveg haanygvngs nsgsnngsng qngsttavda erpnmeiang tinksgpggg 661ngsgsgsgnd mylkrftqre hrvaavikfr qkrkernfgk kvryqsrkrl aeqrprvrgq 721fvrqavqdqq qqgggreaaa dr

Rice Prr7 protein features: Location/Qualifiers

 source   1..742 /organism=″Oryza sativa Japonica Group″/cultivar=″Nipponbare″ /db_xref=″taxon:39947″  Protein   1..742/product=″pseudo-response regulator 37″  Region    65..180/region_name=″REC″ /note=″Signal receiver domain; originally thought tobe unique to bacteria (CheY, OmpR, NtrC, and PhoB), now recentlyidentified in eukaroytes ETR1 Arabidopsis thaliana; this domain receivesthe signal from the sensor partner in a two-component systems; contains;cd00156″ /db_xref=″CDD:29071″  Site order(68..69,114,122,144,163,166..167) /site_type=″active″/db_xref=″CDD:29071″  Site  114 /site_type=″phosphorylation″/db_xref=″CDD:29071″  Site  order(117..118,120..122) /site_type=″other″/note=″intermolecular recognition site″ /db_xref=″CDD:29071″  Site 166..168 /site_type=″other″ /note=″dimerization interface″/db_xref=″CDD:29071″  Region    682..718 /region_name=″CCT″ /note=″CCTmotif. This short motif is found in a number of plant proteins. It isrich in basic amino acids and has been called a CCT motif after Co, Coland Toc1; pfam06203″ /db_xref=″CDD:87043″  CDS   1..742 /gene=″OsPRR37″/coded_by=″AB189039.1:1..2229″

A difference in the expression (gene regulation) of PRR37 in EBA-3 andRTx436 could cause a difference in gene activity corresponding to Ma6vs. ma6. Preliminary assays showed that PRR37 was expressed differentlyin EBA-3 and RTx436. Therefore, ˜800 bp of the promoter regions of PRR37from EBA-3 and RTx436 was sequenced and aligned (FIG. 3). This revealedmany sequence differences including several large deletions/insertionsin the promoter regions of PRR37 in RTx436 compared to EBA-3 (FIG. 3).These differences in sequence may alter the expression of the PRR37alleles and contribute to a difference in flowering phenotype.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Childs et al., Plant Physiol., 116(3):1003-1011, 1998.-   Childs et al., Plant Physiol., 113:611-619, 1997.-   Crasta et al., Mol. Gen. Genet., 262(3):579-588, 1999.-   Craufurd et al., Theor. Appl. Genet., 99:900-911, 1999.-   Feltus et al., Theor. Appl. Genet., 112(7):1295-1305, 2006.-   Hart et al., Theor. Appl. Genet., 103: 1222-1242, 2001-   Ishikawa et al., Plant Cell, 17(12):3326-3336, 2005.-   Kaczorowski and Quail, Plant Cell, 15(11):2654-2665, 2003.-   Klein et al., Plant Genome, 48: S12-22, 2008-   Lin et al., Genetics, 141(1):391-411, 1995.-   McClung, Proc. Natl. Acad. Sci. USA, 103(32):11819-11820, 2006.-   Miller et al., Crop Science, 8:499-502, 1968.-   Nakamichi et al., Plant Cell Physiol., 48(6):822-832, 2007.-   Paterson et al., Proc. Natl. Acad. Sci. USA, 92(13):6127-6131, 1995.-   Quinby and Karper, Amer. J. Botany, 33(9):716-721, 1946.-   Quinby, J. R., Crop Science 6:516-518, 1966-   Quinby, J. R. (1974) Sorghum Improvement and the Genetics of Growth.    Texas A&M University Press.-   Rooney and Aydin, Crop Science, 39;397-400, 1999.-   Rosyara et al., In: Family-based mapping of FHB resistance QTLs in    hexaploid wheat, Proc. Natl. Fusarium Head Blight Forum, Kansas    City, Mo., 2007.-   Takano et al., Plant Cell, 17(12):3311-3325, 2005.-   Turner et al., Science, 310(5750):1031-1034, 2005.

1-26. (canceled)
 27. A method of screening a sorghum plant for a Ma7allele comprising: a) obtaining a sorghum plant; and b) assaying thesorghum plant for a genetic marker genetically linked to the Ma7 allele,wherein said Ma7 allele is located on chromosome 1 between coordinates6545866 to
 8017655. 28-33. (canceled)
 34. The method of claim 27,wherein the genetic marker is selected from the group consisting ofsequence variants revealed by direct sequence analysis, restrictionfragment length polymorphisms (RFLP), isozyme markers, allele specifichybridization (ASH), amplified variable sequences of plant genome,self-sustained sequence replication, simple sequence repeat (SSR) andarbitrary fragment length polymorphisms (AFLP).
 35. The method of claim27, wherein said Ma7 allele is in a gene encoding a polypeptide selectedfrom the group consisting of PhyC, a MADS-box 14 protein and AP1.
 36. Amethod of producing sorghum plant homozygous for a Ma7 allelecomprising: screening according to the method of claim 27 a plurality ofplants from a segregating population, and selecting one or more plantshomozygous for the Ma7 allele.